Flow cytometers are capable of rapid and efficient analysis (e.g. “high-throughput” analysis) of particles flowing in a stream of liquid to provide real time identification of individual particles that is useful in a large number of applications. Many embodiments of flow cytometer use what is referred to as a “sheath fluid” that surrounds and aligns the particles into single file in what may be referred to as a “core diameter” of the flow profile so that they pass through a detection region in a serial fashion. Alternatively, some embodiments of flow cytometer include a single flow profile with channels having a dimension that is substantially similar in size to the dimensions of the particles allowing the particles to easily pass in single file but preventing multiple particles from passing the detector simultaneously. Particles may include cells, single molecules, droplets of fluid immiscible with the fluid composition of the stream, etc.
Flow cytometer embodiments are available to process sample streams at high flow rates (e.g. ≥10 m/s) for high speed applications and slow flow rates (e.g. ≤10 mm/s) for slow speed applications. While high flow rate flow cytometers are common, slow flow rate flow cytometers are less common and are less reliable at delivering a stable, slow sheath flow rate. The problems are typically rooted in pump and fluidic channel systems that are not well suited to both high and low flow rate throughput. The problems of delivering stable sheath flow rate can be even more difficult for flow cytometry methods that use viscous sheath fluids such as oil.
Embodiments of a flow cytometer commonly use “pressure reservoirs” and “regulators” to drive the flow of sheath fluid, which can be made stable but require careful control over temperature and viscosity of the fluids in order to maintain stability of the flow rate. In practice such systems are limited to a small dynamic range of flow rate and are generally restricted to providing either a fast or a slow sheath flow rate but not both. For example, gravimetric reservoirs have been used to produce very stable slow flow rates but are not generally practical for high flow rate systems.
Volumetric delivery methods are also used, embodiments of which may include syringe pumps and/or peristaltic pumps which are typically more robust with respect to fluid temperature and viscosity differences but are limited in dynamic range and are subject to significant pulsatility. Peristaltic pumps in particular are naturally pulsatile and must be used in conjunction with pulse dampening if any level of acceptable stability in flow rate is to be achieved. Syringe pumps may propagate larger pulse waves due to low stepper motor revolution count and large fluid volume displaced per step. Syringe wear, pump wear and fluid containing salt or particles can induce syringe stiction further exacerbating pulsatility of flow. Additionally, analysis is limited by syringe volume and must be paused for refilling. Choice of syringe size is often a compromise between the greater relative pulsation due to larger displacement volume and stiction inherent in large syringes and the higher frequency of filling required for small syringes. Examples of syringe pump pulsatility are described by Li et al. in Lab Chip. 2014 Feb. 21; 14(4):744-9, titled “Syringe-pump-induced fluctuation in all-aqueous microfluidic system implications for flow rate accuracy”, which is hereby incorporated by reference herein in its entirety for all purposes.
Acquiring precise quantitative data in flow cytometry embodiments depend on stable flow rate for sheath flow that deliver particles with consistent velocity and position through the detection region of the flow cytometer. For example, it is particularly important to have a stable flow rate for sheath flow in embodiments of a flow cytometer with more than one spatially separated laser. Variations in particle flow velocity result in differences in transit time of individual particles from laser to laser, and these variations limit the number of events per second that the flow cytometer can accurately record. As the variation in particle flow velocity increases, the likelihood that the flow cytometer introduces error or even misses data from transiting particles increases.
Therefore, it is highly desirable to have flow cytometer embodiments capable of providing a stable flow at both fast and slow flow rates to provide reliable operation for a wide range of applications.